Ornithine carbamyltransferase is an enzyme in a pathway for biosynthesizing arginine, which catalyzes the reaction of forming citrulline by transferring a carbamyl group from carbamylphosphate to ornithine.
Phaseolotoxin produced by a plant-pathogenic microorganism Pseudomonas syringae pv. phaseolicola inhibits ornithine carbamyltransferase activity. Hence, plants infected with this microorganism or plants sprayed with phaseolotoxin are prevented from being supplied with arginine necessary for protein synthesis, and cell growth is terminated and the plant will be withered at last.
The present invention relates to a rice-derived ornithine carbamyltransferase gene which can be utilized in creating a gene coding for plant ornithine carbamyltransferase having resistance to an inhibitory compound such as phaseolotoxin targeting ornithine carbamyltransferase, as well as in efficient production of an enzyme used in screening an inhibitory compound targeting plant ornithine carbamyltransferase.
Pseudomonas syringae pv. phaseolicola is a plant-pathogenic microorganism carrying a gene for ornithine carbamyltransferase having resistance to self-produced phaseolotoxin.
A recombinant plant having this phaseolotoxin-resistant ornithine carbamyltransferase gene introduced thereinto is reported to exhibit resistance to pathogenic Pseudomonas syringae (Hatziloukas, E. and Panopoulos, N. J., J. Bacteriol., 172: 5895-5909, 1992). However, said gene is derived from the plant-pathogenic microorganism and has, therefor, a potential safety problem in practical use. Accordingly, said gene is not applied to edible crops.
On the other hand, an ornithine carbamyltransferase gene derived from plants, particularly from edible crops, is superior in safety as a practical gene to be introduced. If a mutation can be introduced in this ornithine carbamyltransferase gene from edible crops to create phaseolotoxin resistant ornithine carbamyltransferase, it would be possible to develop a practical ornithine carbamyltransferase gene for producing plants resistant to the toxin.
For development of the plant ornithine carbamyltransferase gene mutated so as to have phaseolotoxin resistance, it is necessary to obtain complementary DNA coding for said enzyme from edible crops. Further, it is necessary to establish an expression system, e.g. in microorganisms, for easily evaluating the functions of said resistant gene.
However, plant complementary DNA proven to code for said enzyme, or an expression system for verifying it, still remains unsolved.
The object of the present invention is to isolate complementary DNA coding for ornithine carbamyltransferase derived from a rice plant, necessary for development of a plant ornithine carbamyltransferase gene mutated to have phaseolotoxin resistance and further to establish an expression system in microorganisms for simply evaluating the functions of said gene mutated to have said resistance.
Incidentally, the use of phaseolotoxin as a herbicidal component in utilizing its inhibitory activity on ornithine carbamyltransferase is examined.
Conventionally, evaluation of the herbicidal activity of such compounds has been carried out by observing physiological alternations as a symptom appearing on a plant sprayed with test compounds. Accordingly, the evaluation process of herbicidal acitivity costs a great deal of time and labor including cultivation of test plants.
In order to develop rapid and easy in vitro evaluation system for potential herbicidal compounds, a means of producing a large amount of rice ornithine carbamyltransferase is necessary, for which it is also necessary to obtain complementary DNA coding for said enzyme.